The subject of the invention is compounds implicated in resistance to eutypa dieback in plants as well as agents for combatting this disease, for example in the grapevine.
At present eutypa dieback is the most serious disease of the grapevine. In fact, it is present in vineyards throughout the world; it leads to the premature death of many grapevines of the sensitive varieties and there exist no curative agents. The parasite responsible for eutypa dieback of the grapevine is an ascomycete fungus existing in a perfect form Eutypa lata (Carter M. V., 1955, Apricot gummosis--a new development J. Dep. Agric. South Aust. 59: 178-184) Tul and C.Tul. Syn; E armeniacae Hansf. and Carter) or an imperfect form Libertella blepharis (Samuel G, 1933 "Gummosis" or "Dieback" in apricot trees J. Dep. Agric. South Aust. 36: 979-980) A. L. Smith (syn. Cytosporina sp.).
Eutypa dieback is certainly an ancient disease, even though the pathogenic agent was observed on the grapevine only in 1973 in Australia. In France the disease was identified with certainty by Bolay in 1977 in the region of Languedoc-Roussillon. From 1978, the observations have multiplied in the vineyards of France which show the expansion of eutypa dieback. The reasons for the spread of this dieback disease, which is endemic, have still not been dearly identified to-day; according to certain authors, this progression might be attributable to the modification of certain cultural practices.
The grapevine is not the only perennial ligneous plant which is host to this parasite. In fact, the signs of eutypa dieback have been observed in particular on trees and bushes such as the apricot, black currant, cherry, tamarind, almond, apple . . . .
The disease affects the trunk and the "arms" of the grapevine, sites of development of the mycelium, and the herbaceous parts which, however, never harbour the parasite. The ascospores of the fungus, released by the asci of the perithecia situated on the rind of the contaminated grapevines, penetrate via the wounds resulting from pruning into the lumen of the vessels of the xylem, then germinate at several millimeters from the surface of the wound and colonize the xylem, the cambium and the phloem. The installation of the mycelium in the "wood" of the trunk and the arms of the grapevine leads to the formation of an always well-defined, hard and sectorial, brown necrosis, which has an appearance of dry rot, a sign of a preferential degradation of the cellulose of the wood.
The plants affected by eutypa dieback show typical symptoms in the spring. In the most characteristic cases, the branches are emaciated with short internodes; the leaves of small size, often coiled or even deformed, have a slightly chlorotic appearance and sometimes small marginal necroses. The inflorescences seem to develop normally until flowering, but they may then necrose or give bunches of small grapes (fruit drop). The symptoms described often affect one arm but the disease can then spread to all of the grapevine, leading to the death of the latter.
The duration of the slow incubation of the fungus in the trunk and the arms (3 to 10 years) confers on this disease an insidious character. The expression of the symptoms on the herbaceous organs is uncertain depending on the year: a contaminated grapevine does not show symptoms regularly, certainly for reasons related to environmental factors and in particular to climatic conditions. Moreover, the existence of isolates of different aggressivity has been demonstrated, making still more difficult the recognition of the disease.
The destruction of the parasite in the trunk of the grapevines or the reduction of its expression is not possible to-day. In the absence of curative pest control measures, different prophylactic and preventive measures have been recommended to contain the disease. It is recommended, for example, to limit the pressure of the inoculum by removing old grapevines and the arms cut after the pruning operation. Awareness of the conditions of release and germination of the ascospores has led to several recommendations: prune late while taking account of the climatic conditions (dry and calm weather), avoid prunings causing large surface wounds, protect each pruning wound by the manual application of a fungicide to limit the germination of the ascospores. Finally, the affected strains may be restored by the operation of cutting them back severely, which consists of preserving a stem existing below the diseased part of the trunk and of removing the upper part.
It can be seen that eutypa dieback is certainly harmful because the present pest control agents, uniquely prophylactic and preventive, are restrictive, expensive and of limited scope.
The economic impact of this disease is considerable. The surveys conducted in France since 1988 show that the levels of symptoms are variable and depend on the variety of grapevine and, for a given grapevine, depend on the year and the regions, sometimes up to 50% of the grapevines being subjected to the disease. The most sensitive varieties include the Sauvignon blanc, the Cabernet Sauvignon, the Ugni blanc, the Cinsault and the Chein whereas the Merlot and the Semillon are the most tolerant.
A network of observations based on a rigorous statistical approach set up in the context of a European contract ("Eutypa dieback control" programme No. 8001-CT-91.205) focussed on 85000 grapevines cultivated in 11 wine-growing regions and on 10 varieties of grapevine. It made it possible to show that all the varieties exhibit symptoms of eutpa dieback but to very variable extents: 30 to 80% in certain regions of Greece, 5 to 10% in the Rioja Alta region (Spain, Tempranillo variety) and in Italy (Trebbiano variety) and less than 2%, even 1%, in the wine-growing areas of Portugal.
This disease may also entail a reduction of the expression of the aromatic quality in certain grapevine varieties. In addition, eutypa dieback requires the premature replacement of the dead varieties which causes the rejuvenation of the vineyard, which is detrimental to quality. It should also be noted that the parasitic fungus plays a pioneer role in the installation of parasitic apoplexy (Carignon C., 1991), fungal complex also responsible for a dieback of the grapevine.
This disease also has indirect effects on quality by modifying the planting of the vines in the vineyards because the sensitive varieties are little by little replaced by more tolerant varieties, while however respecting the restrictions of the registered designation of origin. Furthermore, if the harvest volume is maintained in spite of varieties missing to a high extent, the quality of the wines may be affected.
Consequently, it is dear that for the winegrowers wishing to maintain a regular production of quality wines, eutypa dieback is presently the most worrying disease.
The inventors have shown that eutypa dieback is associated with the synthesis of a compound of the family of the lipophilic acids, 4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzaldehyde, called eutypine (Tey-Rulh P. et al. Phytochemistry, vol. 30, No. 2, pp. 471-473, 1991) in some tissues of the host harbouring the parasitic fungus. This toxic molecule is synthesized by the parasitic fungus.
In order to control this disease the inventors have been concerned with the structure and physiology of eutypine when it is synthesized in the host by the parasite and have observed in plants and in particular in the plants resistant to eutypa dieback, the synthesis of degradation products of this toxic constituent.
By the expression "plants resistant to eutypa dieback" is meant in the context of the present application, the capacity of the plants contaminated by the parasite responsible to resist the harmful effects induced directly or indirectly by the presence of the parasite.
This type of resistance may also be explained as the capacity of the plants contaminated by the fungus responsible for eutypa dieback to tolerate the presence of this parasite without their development, including their growth and their physiology, being affected to an extent which would entail the dieback of the plants in question.
The inventors have demonstrated a metabolic pathway for eutypine within the plants contaminated by the parasite, resulting in the synthesis of products non-toxic for these plants; as a result, they have suggested agents for conferring on plants sensitive to eutypa dieback properties of resistance or, where appropriate, agents for increasing the resistance capacities already existing in certain plants towards eutypa dieback.
In this connection, the inventors have identified and characterized in particular a polypeptide with enzymatic activity of the reductase type capable of promoting the degradation of the toxic eutypine into non-toxic constituents including a compound called eutypinol.
The inventors have isolated and identified the eutypine reductase enzyme and have succeeded in characterizing the nucleotide sequence coding for this enzyme.
The nucleotide sequence coding for this enzyme thus constitutes a novel means for considering the production of plants resistant to eutypa dieback in conformity with the definition which was given previously of this resistance. For example, the characterization of this nucleotide sequence makes it possible to prepare transgenic plants capable of overproducing eutypine reductase and of increasing the metabolism of the eutypine produced in the host by the parasite.